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Cd2+ effect on free radicals in Cladosporium cladosporioides-melanin tested by EPR spectroscopy
Chemical Physics Letters 394 (2004) 366–371 www.elsevier.com/locate/cplett
Cd2+ effect on free radicals in Cladosporium cladosporioides-melanin tested by EPR spectroscopy Magdalena Matuszczyk a, Ewa Buszman b, Barbara Pilawa Teresa Witoszyn´ska b, Tadeusz Wilczok c
a,*
,
a Department of Medical Physics, School of Pharmacy, Medical University of Silesia, Jagiellon´ska 4, PL-41-200 Sosnowiec, Poland Department of Pharmaceutical Chemistry, School of Pharmacy, Medical University of Silesia, Jagiellon´ska 4, PL-41-200 Sosnowiec, Poland Department of Molecular Biology and Genetics, School of Pharmacy, Medical University of Silesia, Jagiellon´ska 4, PL-41-200 Sosnowiec, Poland b
c
Received 24 March 2004; in final form 8 July 2004 Available online 30 July 2004
Abstract Changes in free radicals system of Cladosporium cladosporioides-melanin and model DOPA-melanin caused by diamagnetic Cd2+ ions were studied by electron paramagnetic resonance (EPR) spectroscopy. EPR line of eumelanin was mainly found in the spectrum of Cl.cl.-melanin. Cd2+ ions increased o-semiquinone free radicals concentration in both natural and synthetic melanins. Cd2+ broadened EPR lines of Cl.cl.-melanin in mycelium and the ions fastened spin–lattice relaxation processes. The narrower EPR lines and slower spin–lattice relaxation were obtained for DOPA-melanin–Cd2+ complexes than for DOPA-melanin. Pheomelanin additionally existing in Cl.cl. samples was responsible for differences between the EPR data for Cl.cl. melanin and DOPA-melanin. 2004 Elsevier B.V. All rights reserved.
1. Introduction Melanins are known as the paramagnetic biopolymers [1–15]. o-Semiquinone free radicals are responsible for paramagnetism of these structures in human organism and biological systems [1–8,10,15]. Melanins reveal high activity in binding of drugs [10] and metal ions [2–8,10,16]. In this Letter, the complexes of melanin from pigmented soil fungi with diamagnetic cadmium ions were studied. The influence of dia- and paramagnetic metal ions on free radical concentrations in synthetic eu- and pheomelanins is well known [2–8,10,16]. These concentrations increase and decrease in melanins complexes with dia- and paramagnetic metal ions, respectively. It was proposed by Sarna et al. [2–7] that the quench*
Corresponding author. Fax: +48 032 291 74 66. E-mail address: [email protected] (B. Pilawa).
0009-2614/$ - see front matter 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.cplett.2004.07.013
ing of melanin free radical EPR signal by paramagnetic metal ions reveals physical nature. Dipole– dipole interactions between unpaired electrons of free radical and metal ion decrease amplitude of melanin free radicals EPR line. It was shown [7] that this effect increases for the higher concentrations of paramagnetic metal ions and for the faster spin–lattice relaxing metal ions. The aim of this Letter was to examine the effect of diamagnetic Cd2+ ions on paramagnetic properties of complex natural melanin existing in Cl.cl., which is the mixture of eu- and pheomelanin. The Cl.cl.-melanin was chosen for our studies, because of its important role in the environmental protection and high ability to metal ions binding [10,17,18]. The changes in free radical system of melanin biopolymers were analyzed by EPR spectroscopy. The effect of Cd2+ on magnetic interactions in Cl.cl.-melanin was tested.
M. Matuszczyk et al. / Chemical Physics Letters 394 (2004) 366–371
2. Experimental 2.1. Samples The model eumelanin – synthetic DOPA-melanin – was formed by oxidative polymerization of 3,4-dihydroxyphenylalanine (L -DOPA) in 0.07 M phosphate buffer at pH 8.0 [19]. Natural melanin was obtained from dry mycelium Cl.cl. [12]. Fungi were hydrolyzed in 6 M HCl at 110 C for 24 h to remove the protein and washed with bidistilled water. The insoluble melanin was degreased with acetone and dried to a constant weight. The total efficiency of melanin isolation, calculated as the ratio of the amount of obtained melanin divided by the amount of dry mycelium, multiplied by 100, was 6.8%. Cadmium (II)–melanin complexes were obtained as follows: 100 mg of synthetic DOPA-melanin, 100 mg of melanin isolated from Cl.cl., and 100 mg of dry mycelium Cl.Cl. were mixed with 100 ml of metal ion solution containing 1 · 104 M Cd2+. All samples were incubated for 24 h at room temperature and then filtered. The amounts of Cd2+ bound to melanin were determined by the use of atomic absorption spectrophotometer type AAS 3 (Carl Zeiss, Jena). The final metal ion–melanin complexes contained 10.6 lg Cd2+/mg DOPA-melanin, 10.1 lg Cd2+/mg Cl.cl.-melanin and 7.1 lg Cd2+/mg mycelium.
367
g-Factor was calculated from resonance condition: g ¼ hm=bBr ; where h is the Planck constant; m is the microwave frequency; b is the Bohr magnetron; and Br is the resonance magnetic induction. The influence of microwave power on amplitude and linewidth of the resonance absorption curves of DOPAmelanin, Cl.cl.-melanin, mycelium Cl.cl. and their complexes with diamagnetic Cd2+ ions were determined.
3. Results EPR spectra of the investigated melanins are presented in Fig. 1. Single EPR lines were measured for model eumelanin – DOPA-melanin. The complex character of EPR spectra was obtained for melanin isolated from Cl.cl. and for melanin existing in mycelium Cl.cl. EPR
1 mT DOPA-melanin
2.2. EPR measurements EPR measurements for dry melanin samples were performed using an X-band (9.3 GHz) spectrometer with modulation of magnetic field of 100 kHz. The microwave frequency was recorded. EPR spectra were measured with attenuation of 20 dB (0.7 mW) to avoid microwave saturation of resonance absorption curves. The melanin samples were in contact with atmospheric oxygen. Free radical concentration in the samples (N), g-factor and linewidths (DBpp) of EPR spectra, were measured. Ultramarine was used as a reference of concentration of paramagnetic centers. A ruby crystal permanently placed in the spectrometer cavity was the secondary reference. Concentration of paramagnetic centers was calculated according to the formula: N ¼ nu ½ðW u Au Þ=P u ½P =ðWAmÞ; where nu is the number of paramagnetic centers in ultramarine (1.19 · 1019 [spin]); W, Wu is the receiver gain for the sample and ultramarine; A, Au is the amplitude of ruby signal for the sample and ultramarine; P, Pu is the area under the absorption curve for the sample and ultramarine; and m is the mass of the sample. Area under the absorption curve was obtained by double integration of its first derivative using the INTER program.
1 mT Cl.cl.-melanin
1 mT mycelium Cl.cl.
Fig. 1. EPR spectra of DOPA-melanin, melanin isolated from Cladosporium cladosporioides and melanin existing in mycelium Cl.cl.
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Table 1 Free radicals concentration (N) and EPR spectra parameters (DBpp, g-factor) of synthetic DOPA-melanin, natural melanin from Cladosporium cladosporioides and their complexes with Cd2+ Sample
N (spin/g)
DBpp (mT) ± 0.02
g ± 0.0002
DOPA-melanin DOPA-melanin + Cd2+ Cl.cl.-melanin Cl.cl.-melanin + Cd2+ Mycelium Cl.cl. Mycelium Cl.cl. + Cd2+
2.5 · 1019 2.9 · 1019 4.1 · 1018 1.6 · 1019 9.3 · 1016 4.4 · 1018
0.48 0.41 0.32 0.34 0.32 0.38
2.0043 2.0042 2.0040 2.0041 2.0042 2.0041
EPR spectra were recorded at microwave power 0.7 mW.
spectra of both Cl.cl. samples were superposition of eumelanin single line and pheomelanin line with unresolved hyperfine structure. The complex character of these EPR lines was more visible for the spectra recorded at higher microwave powers. The single line of eumelanin was the main component in the experimental spectra of Cl.cl.-melanin and mycelium Cl.cl. The additional pheomelanin component in the EPR spectra of two studied Cl.cl.-melanin samples revealed only low amplitude. Diamagnetic Cd2+ ions did not change lineshapes of melanin EPR spectra. Single and complex EPR lines were measured for the analyzed melanins and their complexes with cadmium. Concentrations of free radicals N [spin/g], and parameters of EPR spectra (linewidths DBpp and g-factors) of DOPA-melanin, melanin isolated from Cl.cl. and mycelium Cl.cl. are presented in Table 1.
Free radicals with similar g values of 2.0040–2.0043 exist in the studied samples. The highest concentration of free radicals (2.5 · 1019 spin/g) was obtained for the model DOPA-melanin, while the lowest concentration (4.1 · 1018 spin/g) was measured for natural melanin isolated from Cl.cl. (Table 1). The free radical concentration in mycelium Cl.cl. was only 9.3 · 1016 spin/g. Diamagnetic Cd2+ ions increase the concentration of free radicals in all melanin samples studied. The strongest changes of free radicals concentration caused by cadmium ions were observed for mycelium Cl.cl. The broad EPR lines with linewidths DBpp in the range 0.32–0.48 mT were measured for both synthetic and natural melanin samples (Table 1). The relatively narrower (DBpp = 0.32 mT) EPR lines were recorded for natural melanin samples. Cd2+ ions decreased the EPR linewidth of DOPA-melanin (by 0.07 mT), but they broadened EPR lines of mycelium Cl.cl. (by 0.06
1.2 2
1
0.8 AMPLITUDE
AMPLITUDE
1.5
1
0.6
0.4 0.5
0.2
DOPA-melanin DOPA-melanin+Cd2+
Cl.cl.-melanin
0 0
0.2
0.4
0.6
0.8
1
1/2
(M/Mo)
Cl.cl.-melanin+Cd2+
0 0
0.2
0.4
0.6
0.8
1
1/2
(M/Mo) Fig. 2. Influence of microwave power on amplitudes of EPR spectra of DOPA-melanin and DOPA-melanin + Cd2+ complexes. Mo, M – total microwave power produced by klystron and used microwave power, respectively.
Fig. 3. Influence of microwave power on amplitudes of EPR spectra of Cl.cl.-melanin and Cl.cl.-melanin + Cd2+ complexes. Notations as in Fig. 2.
M. Matuszczyk et al. / Chemical Physics Letters 394 (2004) 366–371
0.03
0.025
AMPLITUDE
0.02
0.015
0.01
0.005 mycelium Cl.cl.
mycelium Cl.cl.+Cd2+
0 0
0.2
0.4
0.6
0.8
1
1/2
(M/Mo)
Fig. 4. Influence of microwave power on amplitudes of EPR spectra of mycelium Cl.cl. and mycelium Cl.cl. + Cd2+ complexes. Notations as in Fig. 2.
mT). The EPR linewidths of Cl.cl.-melanin and its complexes with Cd2+ were similar. The influence of microwave power on the amplitudes and linewidths of EPR spectra of DOPA-melanin, melanin isolated from Cl.cl. and mycelium Cl.cl. are presented in Figs. 2-5a, 3-5b and 4-5c, respectively. Amplitudes of the all analyzed samples increased with increasing of microwave power, reached the maximum values and then decreased with microwave power (Figs. 2–4). The linewidths increased with increasing of microwave powers (Fig. 5). Cd2+ ions complexed with melanins did not change the character of the above correlations. However, in the presence of Cd2+ the maxima in the correlations presented in Figs. 2–4 shifted to lower and higher values of microwave powers for DOPA-melanin and Cl.cl. samples, respectively.
369
The obtained results confirmed the fact that diamagnetic Cd2+ ions increase the concentration of o-semiquinone free radicals in melanin (Table 1). This effect was observed for synthetic and natural melanins earlier [2,10,20]. The increase of free radical concentrations was measured for melanin complexes with Cd2+, Zn2+, Al3+, La3+ and Sc3+ [20]. In this Letter, we demonstrated that cadmium ions strongly change the free radical concentration in melanin existing in mycelium Cl.cl. It seems that the observed differences in changes of free radical concentrations in melanin (DOPA- and Cl.cl.-melanin) and mycelium samples are rather not caused by different Cd2+ amounts bound to melanin. In mycelium Cl.cl., Cd2+ ions can be bound not only to melanin biopolymers but also to proteins, carbohydrates and other components of mycelium. The ability of melanin to bind metal ions depends on the type of sample, but diamagnetic cadmium ions rise only 1,2 and 3,9 times the free radical concentration in DOPA-melanin and Cl.cl.-melanin, respectively, and as many as 47.3 times in mycelium. We propose that the strong influence of cadmium ions on free radical concentration in melanin existing in mycelium Cl.cl. reveals rather physical character and probably is not related to Cd2+ coordination to melanin radicals. It seems to be acceptable that magnetic interactions between paramagnetic melanin–Cd2+ complexes and low amounts of paramagnetic centers of other constituents of mycelium are one of the reasons for this effect, however, it remains unresolved now. By the first time, it was proved that even a low amount of pheomelanin in the sample, containing mainly eumelanin, strongly changed magnetic interactions in the sample. Cd2+ ions shortened spin–lattice relaxation time in Cl.cl.-melanin. It is proposed that cross-relaxation processes appear in Cl.cl.-melanin–Cd2+ complexes. Probably, the cross-relaxation processes are induced by pheomelanin. This point of view is confirmed by the absence of such effects in DOPA-melanin–Cd2+ complexes.
5. Conclusions
4. Discussion EPR spectroscopy was used to determine the melanin type in Cl.cl. The shape of EPR spectra of Cl.cl.-melanin indicated that the studied biopolymer is a mixture of euand pheomelanin, but mainly eumelanin exists in Cl.cl. samples and only a low amount of pheomelanin was identified. The EPR signal of pheomelanin was stronger for Cl.cl. samples measured at higher microwave powers. Dipolar broadened EPR lines of eumelanin dominated in the resonance absorption spectra recorded at all microwave powers.
The existence of a low amount of pheomelanin, besides eumelanin, in the Cl.cl. samples was proved by the analysis of shape of their EPR spectra. Diamagnetic Cd2+ ions increased the concentration of o-semiquinone free radicals in both model DOPA-melanin and Cl.cl.melanin. The various Cd2+ effects on magnetic interactions in synthetic eumelanin and in the analyzed natural biopolymer were shown by the use of EPR spectroscopy. The stronger dipolar interactions between paramagnetic centers exist in complexes of mycelium Cl.cl. with Cd2+ than in the crude mycelium. Faster spin–lattice relaxation processes appeared in Cl.cl.-melanin and in mycelium Cl.cl. after complexing with Cd2+.
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M. Matuszczyk et al. / Chemical Physics Letters 394 (2004) 366–371
∆ B pp [mT]
(a) 0.6
0.5
0.4 DOPA-melanin
DOPA-melanin+Cd2+
0.3 0
0.2
0.4
1/2
(M/M o)
0.6
0.8
1
∆ B pp [mT]
(b) 0.42
0.38
0.34 Cl.cl.-melanin
Cl.cl.-melanin+Cd2+
0.3 0
0.2
0.4
1/2
0.6
0.8
1
(M/M o)
∆ B pp [mT]
(c) 0.45
0.4
0.35 mycelium Cl.cl.
mycelium Cl.cl.+Cd2+
0.3 0
0.2
0.4
0.6
0.8
1
1/2
(M/M o)
Fig. 5. Influence of microwave power on linewidths DBpp of EPR spectra of: (a) DOPA-melanin and DOPA-melanin + Cd2+ complexes, (b) Cl.cl.melanin and Cl.cl.-melanin + Cd2+ complexes, and (c) mycelium Cl.cl. and mycelium Cl.cl. + Cd2+ complexes. Notations as in Fig. 2.
It is proposed that spin–lattice interactions in DOPAmelanin (eumelanin) and Cl.cl.-melanin (the mixture of eu- and pheomelanin) are different. The decrease of dipolar spin–spin interactions in complexes of DOPAmelanin with Cd2+ was observed. Diamagnetic Cd2+ ions did not significantly change spin–spin interactions in Cl.cl.-melanin.
Acknowledgement This work was financially supported by the Medical University of Silesia. References [1] R.C. Sealy, J.S. Hyde, C.C. Felix, I.A. Menon, G. Prota, Science 217 (1982) 545. [2] T. Sarna, J.S. Hyde, H.M. Swarz, Science 192 (1976) 1132.
[3] T. Sarna, W. Froncisz, J.S. Hyde, Arch. Biochem. Biophys. 202 (1980) 304. [4] T. Sarna, W. Korytowski, M. Pasenkiewicz-Gierula, E. Gudowska, in: M. Seiji (Ed.), Proceedings of the XI International Pigment Cell Conference, Sendai, Japan, 1980, University of Tokyo Press, Japan, 1981, p. 23. [5] R.C. Sealy, C.C. Felix, J.S. Hyde, H.M. Swartz, in: W.A. Pryor (Ed.), Free Radicals in Biology, Academic Press, New York, 1980, p. 209. [6] W. Froncisz, T. Sarna, J.S. Hyde, Arch. Biochem. Biophys. 202 (1980) 289. [7] T. Sarna, J. Photochem. Photobiol. B: Biol. 12 (1992) 215. [8] M.M. Jastrzezbska, H. Isotalo, J. Paloheimo, H. Stubb, B. Pilawa, J. Biomater. Sci. Polym. Ed. 7 (1996) 781. [9] M. Okazaki, K. Kuwata, Y. Miki, S. Shiga, T. Shiga, Arch. Biochem. Biophys. 242 (1985) 197. [10] E. Buszman, Habilitation Thesis, Medical University of Silesia, Katowice, 1994 (in Polish). [11] E. Buszman, A. Wiewio´ra, B. Bilin´ska, Post. Fiz. Med. 26 (1991) 3 (in Polish). [12] E. Buszman, A. Wiewio´ra, J. Andrzejczyk, T. Wilczok, Curr. Top. Biophys. 16 (1992) 73. [13] E. Buszman, M. Latocha, B. Pilawa, T. Wilczok, Pol. J. Med. Phys. Eng. 1 (1995) 121.
M. Matuszczyk et al. / Chemical Physics Letters 394 (2004) 366–371 [14] E. Buszman, B. Pilawa, M. Latocha, T. Wilczok, D. TyrawskaSpychałowa, C. Bajan, A. Bijak, B. Bilin´ska, Curr. Top. Biophys. 22 (1998) 21. [15] B. Pilawa, E. Buszman, M. Latocha, T. Wilczok, Pol. J. Med. Phys. Eng. 2 (1996) 59. [16] S.Z. Senior, L.L. Mans, H.D. VanGuilder, K.A. Kelly, M.P. Hendrich, T.E. Elgren, Biochemistry 42 (2003) 4392.
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[17] R.A. Nicolaus, in: E. Lederer (Ed.), Ser. Chemistry of Natural Products, Hermann, Paris, 1968. [18] D.H. Ellis, D.A. Griffiths, Can. J. Microbiol. 20 (1974) 1379. [19] F. Binns, R.F. Chapman, N.C. Robson, G.A. Swan, A. Waggott, J. Chem. Soc. C (1970) 1128. [20] C.C. Felix, J.S. Hyde, T. Sarna, R.C. Sealy, J. Am. Chem. Soc. 100 (1978) 3922.
Cd2+ effect on free radicals in Cladosporium cladosporioides-melanin tested by EPR spectroscopy Magdalena Matuszczyk a, Ewa Buszman b, Barbara Pilawa Teresa Witoszyn´ska b, Tadeusz Wilczok c
a,*
,
a Department of Medical Physics, School of Pharmacy, Medical University of Silesia, Jagiellon´ska 4, PL-41-200 Sosnowiec, Poland Department of Pharmaceutical Chemistry, School of Pharmacy, Medical University of Silesia, Jagiellon´ska 4, PL-41-200 Sosnowiec, Poland Department of Molecular Biology and Genetics, School of Pharmacy, Medical University of Silesia, Jagiellon´ska 4, PL-41-200 Sosnowiec, Poland b
c
Received 24 March 2004; in final form 8 July 2004 Available online 30 July 2004
Abstract Changes in free radicals system of Cladosporium cladosporioides-melanin and model DOPA-melanin caused by diamagnetic Cd2+ ions were studied by electron paramagnetic resonance (EPR) spectroscopy. EPR line of eumelanin was mainly found in the spectrum of Cl.cl.-melanin. Cd2+ ions increased o-semiquinone free radicals concentration in both natural and synthetic melanins. Cd2+ broadened EPR lines of Cl.cl.-melanin in mycelium and the ions fastened spin–lattice relaxation processes. The narrower EPR lines and slower spin–lattice relaxation were obtained for DOPA-melanin–Cd2+ complexes than for DOPA-melanin. Pheomelanin additionally existing in Cl.cl. samples was responsible for differences between the EPR data for Cl.cl. melanin and DOPA-melanin. 2004 Elsevier B.V. All rights reserved.
1. Introduction Melanins are known as the paramagnetic biopolymers [1–15]. o-Semiquinone free radicals are responsible for paramagnetism of these structures in human organism and biological systems [1–8,10,15]. Melanins reveal high activity in binding of drugs [10] and metal ions [2–8,10,16]. In this Letter, the complexes of melanin from pigmented soil fungi with diamagnetic cadmium ions were studied. The influence of dia- and paramagnetic metal ions on free radical concentrations in synthetic eu- and pheomelanins is well known [2–8,10,16]. These concentrations increase and decrease in melanins complexes with dia- and paramagnetic metal ions, respectively. It was proposed by Sarna et al. [2–7] that the quench*
Corresponding author. Fax: +48 032 291 74 66. E-mail address: [email protected] (B. Pilawa).
0009-2614/$ - see front matter 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.cplett.2004.07.013
ing of melanin free radical EPR signal by paramagnetic metal ions reveals physical nature. Dipole– dipole interactions between unpaired electrons of free radical and metal ion decrease amplitude of melanin free radicals EPR line. It was shown [7] that this effect increases for the higher concentrations of paramagnetic metal ions and for the faster spin–lattice relaxing metal ions. The aim of this Letter was to examine the effect of diamagnetic Cd2+ ions on paramagnetic properties of complex natural melanin existing in Cl.cl., which is the mixture of eu- and pheomelanin. The Cl.cl.-melanin was chosen for our studies, because of its important role in the environmental protection and high ability to metal ions binding [10,17,18]. The changes in free radical system of melanin biopolymers were analyzed by EPR spectroscopy. The effect of Cd2+ on magnetic interactions in Cl.cl.-melanin was tested.
M. Matuszczyk et al. / Chemical Physics Letters 394 (2004) 366–371
2. Experimental 2.1. Samples The model eumelanin – synthetic DOPA-melanin – was formed by oxidative polymerization of 3,4-dihydroxyphenylalanine (L -DOPA) in 0.07 M phosphate buffer at pH 8.0 [19]. Natural melanin was obtained from dry mycelium Cl.cl. [12]. Fungi were hydrolyzed in 6 M HCl at 110 C for 24 h to remove the protein and washed with bidistilled water. The insoluble melanin was degreased with acetone and dried to a constant weight. The total efficiency of melanin isolation, calculated as the ratio of the amount of obtained melanin divided by the amount of dry mycelium, multiplied by 100, was 6.8%. Cadmium (II)–melanin complexes were obtained as follows: 100 mg of synthetic DOPA-melanin, 100 mg of melanin isolated from Cl.cl., and 100 mg of dry mycelium Cl.Cl. were mixed with 100 ml of metal ion solution containing 1 · 104 M Cd2+. All samples were incubated for 24 h at room temperature and then filtered. The amounts of Cd2+ bound to melanin were determined by the use of atomic absorption spectrophotometer type AAS 3 (Carl Zeiss, Jena). The final metal ion–melanin complexes contained 10.6 lg Cd2+/mg DOPA-melanin, 10.1 lg Cd2+/mg Cl.cl.-melanin and 7.1 lg Cd2+/mg mycelium.
367
g-Factor was calculated from resonance condition: g ¼ hm=bBr ; where h is the Planck constant; m is the microwave frequency; b is the Bohr magnetron; and Br is the resonance magnetic induction. The influence of microwave power on amplitude and linewidth of the resonance absorption curves of DOPAmelanin, Cl.cl.-melanin, mycelium Cl.cl. and their complexes with diamagnetic Cd2+ ions were determined.
3. Results EPR spectra of the investigated melanins are presented in Fig. 1. Single EPR lines were measured for model eumelanin – DOPA-melanin. The complex character of EPR spectra was obtained for melanin isolated from Cl.cl. and for melanin existing in mycelium Cl.cl. EPR
1 mT DOPA-melanin
2.2. EPR measurements EPR measurements for dry melanin samples were performed using an X-band (9.3 GHz) spectrometer with modulation of magnetic field of 100 kHz. The microwave frequency was recorded. EPR spectra were measured with attenuation of 20 dB (0.7 mW) to avoid microwave saturation of resonance absorption curves. The melanin samples were in contact with atmospheric oxygen. Free radical concentration in the samples (N), g-factor and linewidths (DBpp) of EPR spectra, were measured. Ultramarine was used as a reference of concentration of paramagnetic centers. A ruby crystal permanently placed in the spectrometer cavity was the secondary reference. Concentration of paramagnetic centers was calculated according to the formula: N ¼ nu ½ðW u Au Þ=P u ½P =ðWAmÞ; where nu is the number of paramagnetic centers in ultramarine (1.19 · 1019 [spin]); W, Wu is the receiver gain for the sample and ultramarine; A, Au is the amplitude of ruby signal for the sample and ultramarine; P, Pu is the area under the absorption curve for the sample and ultramarine; and m is the mass of the sample. Area under the absorption curve was obtained by double integration of its first derivative using the INTER program.
1 mT Cl.cl.-melanin
1 mT mycelium Cl.cl.
Fig. 1. EPR spectra of DOPA-melanin, melanin isolated from Cladosporium cladosporioides and melanin existing in mycelium Cl.cl.
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M. Matuszczyk et al. / Chemical Physics Letters 394 (2004) 366–371
Table 1 Free radicals concentration (N) and EPR spectra parameters (DBpp, g-factor) of synthetic DOPA-melanin, natural melanin from Cladosporium cladosporioides and their complexes with Cd2+ Sample
N (spin/g)
DBpp (mT) ± 0.02
g ± 0.0002
DOPA-melanin DOPA-melanin + Cd2+ Cl.cl.-melanin Cl.cl.-melanin + Cd2+ Mycelium Cl.cl. Mycelium Cl.cl. + Cd2+
2.5 · 1019 2.9 · 1019 4.1 · 1018 1.6 · 1019 9.3 · 1016 4.4 · 1018
0.48 0.41 0.32 0.34 0.32 0.38
2.0043 2.0042 2.0040 2.0041 2.0042 2.0041
EPR spectra were recorded at microwave power 0.7 mW.
spectra of both Cl.cl. samples were superposition of eumelanin single line and pheomelanin line with unresolved hyperfine structure. The complex character of these EPR lines was more visible for the spectra recorded at higher microwave powers. The single line of eumelanin was the main component in the experimental spectra of Cl.cl.-melanin and mycelium Cl.cl. The additional pheomelanin component in the EPR spectra of two studied Cl.cl.-melanin samples revealed only low amplitude. Diamagnetic Cd2+ ions did not change lineshapes of melanin EPR spectra. Single and complex EPR lines were measured for the analyzed melanins and their complexes with cadmium. Concentrations of free radicals N [spin/g], and parameters of EPR spectra (linewidths DBpp and g-factors) of DOPA-melanin, melanin isolated from Cl.cl. and mycelium Cl.cl. are presented in Table 1.
Free radicals with similar g values of 2.0040–2.0043 exist in the studied samples. The highest concentration of free radicals (2.5 · 1019 spin/g) was obtained for the model DOPA-melanin, while the lowest concentration (4.1 · 1018 spin/g) was measured for natural melanin isolated from Cl.cl. (Table 1). The free radical concentration in mycelium Cl.cl. was only 9.3 · 1016 spin/g. Diamagnetic Cd2+ ions increase the concentration of free radicals in all melanin samples studied. The strongest changes of free radicals concentration caused by cadmium ions were observed for mycelium Cl.cl. The broad EPR lines with linewidths DBpp in the range 0.32–0.48 mT were measured for both synthetic and natural melanin samples (Table 1). The relatively narrower (DBpp = 0.32 mT) EPR lines were recorded for natural melanin samples. Cd2+ ions decreased the EPR linewidth of DOPA-melanin (by 0.07 mT), but they broadened EPR lines of mycelium Cl.cl. (by 0.06
1.2 2
1
0.8 AMPLITUDE
AMPLITUDE
1.5
1
0.6
0.4 0.5
0.2
DOPA-melanin DOPA-melanin+Cd2+
Cl.cl.-melanin
0 0
0.2
0.4
0.6
0.8
1
1/2
(M/Mo)
Cl.cl.-melanin+Cd2+
0 0
0.2
0.4
0.6
0.8
1
1/2
(M/Mo) Fig. 2. Influence of microwave power on amplitudes of EPR spectra of DOPA-melanin and DOPA-melanin + Cd2+ complexes. Mo, M – total microwave power produced by klystron and used microwave power, respectively.
Fig. 3. Influence of microwave power on amplitudes of EPR spectra of Cl.cl.-melanin and Cl.cl.-melanin + Cd2+ complexes. Notations as in Fig. 2.
M. Matuszczyk et al. / Chemical Physics Letters 394 (2004) 366–371
0.03
0.025
AMPLITUDE
0.02
0.015
0.01
0.005 mycelium Cl.cl.
mycelium Cl.cl.+Cd2+
0 0
0.2
0.4
0.6
0.8
1
1/2
(M/Mo)
Fig. 4. Influence of microwave power on amplitudes of EPR spectra of mycelium Cl.cl. and mycelium Cl.cl. + Cd2+ complexes. Notations as in Fig. 2.
mT). The EPR linewidths of Cl.cl.-melanin and its complexes with Cd2+ were similar. The influence of microwave power on the amplitudes and linewidths of EPR spectra of DOPA-melanin, melanin isolated from Cl.cl. and mycelium Cl.cl. are presented in Figs. 2-5a, 3-5b and 4-5c, respectively. Amplitudes of the all analyzed samples increased with increasing of microwave power, reached the maximum values and then decreased with microwave power (Figs. 2–4). The linewidths increased with increasing of microwave powers (Fig. 5). Cd2+ ions complexed with melanins did not change the character of the above correlations. However, in the presence of Cd2+ the maxima in the correlations presented in Figs. 2–4 shifted to lower and higher values of microwave powers for DOPA-melanin and Cl.cl. samples, respectively.
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The obtained results confirmed the fact that diamagnetic Cd2+ ions increase the concentration of o-semiquinone free radicals in melanin (Table 1). This effect was observed for synthetic and natural melanins earlier [2,10,20]. The increase of free radical concentrations was measured for melanin complexes with Cd2+, Zn2+, Al3+, La3+ and Sc3+ [20]. In this Letter, we demonstrated that cadmium ions strongly change the free radical concentration in melanin existing in mycelium Cl.cl. It seems that the observed differences in changes of free radical concentrations in melanin (DOPA- and Cl.cl.-melanin) and mycelium samples are rather not caused by different Cd2+ amounts bound to melanin. In mycelium Cl.cl., Cd2+ ions can be bound not only to melanin biopolymers but also to proteins, carbohydrates and other components of mycelium. The ability of melanin to bind metal ions depends on the type of sample, but diamagnetic cadmium ions rise only 1,2 and 3,9 times the free radical concentration in DOPA-melanin and Cl.cl.-melanin, respectively, and as many as 47.3 times in mycelium. We propose that the strong influence of cadmium ions on free radical concentration in melanin existing in mycelium Cl.cl. reveals rather physical character and probably is not related to Cd2+ coordination to melanin radicals. It seems to be acceptable that magnetic interactions between paramagnetic melanin–Cd2+ complexes and low amounts of paramagnetic centers of other constituents of mycelium are one of the reasons for this effect, however, it remains unresolved now. By the first time, it was proved that even a low amount of pheomelanin in the sample, containing mainly eumelanin, strongly changed magnetic interactions in the sample. Cd2+ ions shortened spin–lattice relaxation time in Cl.cl.-melanin. It is proposed that cross-relaxation processes appear in Cl.cl.-melanin–Cd2+ complexes. Probably, the cross-relaxation processes are induced by pheomelanin. This point of view is confirmed by the absence of such effects in DOPA-melanin–Cd2+ complexes.
5. Conclusions
4. Discussion EPR spectroscopy was used to determine the melanin type in Cl.cl. The shape of EPR spectra of Cl.cl.-melanin indicated that the studied biopolymer is a mixture of euand pheomelanin, but mainly eumelanin exists in Cl.cl. samples and only a low amount of pheomelanin was identified. The EPR signal of pheomelanin was stronger for Cl.cl. samples measured at higher microwave powers. Dipolar broadened EPR lines of eumelanin dominated in the resonance absorption spectra recorded at all microwave powers.
The existence of a low amount of pheomelanin, besides eumelanin, in the Cl.cl. samples was proved by the analysis of shape of their EPR spectra. Diamagnetic Cd2+ ions increased the concentration of o-semiquinone free radicals in both model DOPA-melanin and Cl.cl.melanin. The various Cd2+ effects on magnetic interactions in synthetic eumelanin and in the analyzed natural biopolymer were shown by the use of EPR spectroscopy. The stronger dipolar interactions between paramagnetic centers exist in complexes of mycelium Cl.cl. with Cd2+ than in the crude mycelium. Faster spin–lattice relaxation processes appeared in Cl.cl.-melanin and in mycelium Cl.cl. after complexing with Cd2+.
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∆ B pp [mT]
(a) 0.6
0.5
0.4 DOPA-melanin
DOPA-melanin+Cd2+
0.3 0
0.2
0.4
1/2
(M/M o)
0.6
0.8
1
∆ B pp [mT]
(b) 0.42
0.38
0.34 Cl.cl.-melanin
Cl.cl.-melanin+Cd2+
0.3 0
0.2
0.4
1/2
0.6
0.8
1
(M/M o)
∆ B pp [mT]
(c) 0.45
0.4
0.35 mycelium Cl.cl.
mycelium Cl.cl.+Cd2+
0.3 0
0.2
0.4
0.6
0.8
1
1/2
(M/M o)
Fig. 5. Influence of microwave power on linewidths DBpp of EPR spectra of: (a) DOPA-melanin and DOPA-melanin + Cd2+ complexes, (b) Cl.cl.melanin and Cl.cl.-melanin + Cd2+ complexes, and (c) mycelium Cl.cl. and mycelium Cl.cl. + Cd2+ complexes. Notations as in Fig. 2.
It is proposed that spin–lattice interactions in DOPAmelanin (eumelanin) and Cl.cl.-melanin (the mixture of eu- and pheomelanin) are different. The decrease of dipolar spin–spin interactions in complexes of DOPAmelanin with Cd2+ was observed. Diamagnetic Cd2+ ions did not significantly change spin–spin interactions in Cl.cl.-melanin.
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